Dengue fever is a viral disease transmitted by arthropods most widespread affecting human population. Each year they are reported between 50 and 100 million cases of Dengue, 500 000 of them result in the most severe form of the disease, known as Dengue hemorrhagic fever (Guzman et al., Lancet Infect. Dis. 2002; 2:33-42). The causal agent of this disease is the DV, belonging to the family Flaviviridae, genus flavivirus. DV is a viral complex comprising four serotypes. It is an enveloped virus whose lipid membrane contains two of its three structural proteins: the envelope protein and membrane protein. This lipoprotein envelope surrounds the icosahedral nucleocapsid composed by the third of their structural proteins, the capsid protein. (Leyssen et al., Clin. Microbiol. Rev. 2000; 13:67-82).
In the recent decades, the global spread of infections with these viruses has made the development of an effective vaccine, a public health priority. This purpose has been limited by several factors. First of all, infection with one serotype does not induce long-lasting cross protection against the remaining serotypes (Leyssen et al., Clin. Microbiol. Rev. 2000; 13:67-82) and, at the same time, heterotypic secondary infections are the main risk factor for the development of severe forms of the disease (Guzman et al., Lancet Infect. Dis. 2002; 2:33-42; Mongkolsapaya et al., Nat. Med. 2003; 9:921-927). Therefore, an ideal vaccine against DV should induce a long lasting protective immunity against the four viral serotypes (DV1, DV2, DV3 and DV4).
The most advanced vaccine candidates are based on attenuated viral strains through serial passages in cell cultures, or obtained by recombinant way. The viral interference, among the four serotypes in the tetravalent formulations, is the main limitation of this type of candidates making difficult to induce an equivalent functional immune response against the four serotypes; furthermore, they require to be administrated at long intervals between the two or three vaccine doses proposed. (Bhamarapravati et al., Vaccine. 2000; 18:44-47; Kanesa-Thasan et al., Vaccine. 2001; 19:3179-3188; Morrison et al., J. Infect. Dis. 2010; 201:370-377). In addition, due to their nature as live viruses, they cannot be administrated in children less than one year of age.
As an attractive alternative, a series of preclinical studies based on subunit vaccines have been developed. This approach has three key advantages over vaccination with live attenuated virus: 1) they are potentially safe vaccines, 2) the phenomenon of viral interference should not occur due to the non-replicative nature of the immunogen and 3) Short vaccination schemes can be proposed, contrary to the administrations of live attenuated virus which require long intervals between vaccine doses to achieve the booster effect.
One of the most promising subunit vaccine candidates, is developed by the company Hawaii Biotech/Merck (Hombach, Rev. Panam. Salud Publica. 2007; 21:254-260). It is a candidate formed by each viral envelope protein from the four serotypes, expressed in insect cells. Monovalent and tetravalent formulations have been assessed in mice and monkeys with immunogenicity results similar to those obtained with the attenuated viruses (Clements et al., Vaccine. 2010; 28:2705-2715). Nevertheless, the monovalent formulations required the addition of potent adjuvants, not licensed for human use, to induce a proper immune response. In turn, the tetravalent formulation assessed in non-human primates contained, not only a non-licensed adjuvant, but also the protein NS1 from DV2, which has some homology with endothelial human cells and consequently, it could provoke an autoimmunity disorder. Additionally, there are no data available about the induction of cell-mediated immunity upon administration of this vaccine candidate, an important arm of the immunity, which has been recently identified as having a protective role against dengue. (Gil et al., Viral Immunol. 2009; 22:23-30; Yauch et al., J. Immunol. 2009; 182:4865-4873; Yauch et al., J. Immunol. 2010; 185:5405-5416).
Keeping the advantages associated with the subunit vaccines, and, at the same time, looking for safer immunogenic formulations containing alum as base adjuvant, the group of Cuban researchers has developed a working line based on the capsid protein and the domain III of the envelope protein of dengue virus (Guzman et al., Exp. Rev. Vaccines. 2010; 9:137-147).
The capsid protein from DV is essential in the virion assembly and protects the viral genome being its main function. Its molecular weight is 9-12 kDa (112-127 amino acids) and it has a basic structure since the 25% of its amino acids are Arginine and Lysine. The protein is located within the virion structure, without exposed regions (Kuhn et al., Cell. 2002; 108:717-725), making it attractive to be included into a vaccine, due to it may not be target of immune-enhancer antibodies. On the other hand, various human CTL epitopes have been identified on its sequence, providing the induction of an effective cell-mediated immunity against the virus (Gagnon et al., J. Virol. 1996; 70:141-147; Gagnon et al., J. Virol. 1999; 73:3623-3629).
Although there are several studies on the structural characteristics of this capsid protein, it was not until the year 2007 that it was evaluated for the first time in terms of immunogenicity in mice. In this study, the capsid from DV2, was obtained as recombinant protein in Escherichia coli. Upon a semi purification process, the resultant preparation was assessed in mice, and partial protection after DV2 challenge was obtained without induction of neutralizing antibodies. (Lazo et al., Vaccine. 2007; 25:1064-1070). Later on, purification and in vitro aggregation process was established at lab scale, and again, the resultant protein was assessed in mice to measure its functionality in terms of protection (Lopez et al., Arch. Virol. 2009; 154:695-698). The analysis of immunogenicity revealed the induction of cell-mediated immunity measured by secretion of gamma interferon (IFN-γ), by the splenocytes of mice receiving the aggregated protein. Such a secretion was dependent on CD4+ and CD8+ cells. In turn, upon challenge with DV2, a significant protection was obtained in animals immunized with the aggregated protein and such a protection was also dependent on CD4+ and CD8+ cells (Gil et al., Int. Immunol. 2009; 21:1175-1183). Based on the aforementioned results, it was proposed to combine, in the same genetic construct, the capsid protein and the DomIII region of the envelope protein, both from DV2. DomIII has been widely described as one receptor-binding region (Chen et al., J. Virol. 1996; 70:8765-8772) and, additionally, it has been reported the induction of neutralizing antibodies and protection in mice immunized with fusion proteins containing this viral region. (Crill et al., J. Virol. 2001; 75:7769-7773; Hermida et al., J. Virol. Methods. 2004; 115:41-49; Simmons et al., Am. J. Trop. Med. Hyg. 2001; 65:159-161). In turn, in non-human primates experiments, it has been demonstrated the induction of a protective immune response only using the Freund's adjuvant (Hermida et al., Vaccine. 2006; 24:3165-3171).
The union viral capsid and the DomIII of the viral envelope protein allows the presence of the two regions potentially protective in a same molecule, capable of simultaneously inducing neutralizing antibodies (DomIII) and cellular immune response (capsid). It was then obtained the genetic construct named DIIIC-2 (DomIII fused to the N-terminus region of the capsid protein, serotype 2), which was expressed in E. coli; and the resulting protein was purified at lab scale, and underwent the process of aggregation with a mixture of oligonucleotides of unknown sequence. Upon inoculation of three doses in mice, antiviral and neutralizing antibodies were detected. In a similar way, significant IFN-γ secretion was detected in splenocytes from animals immunized with the aggregated protein. Consistently with the cell-mediated immunity, a significant protection upon intracranial challenge was obtained, and such a protection was mediated by CD4+ and CD8+ cells induced during the immunization process (Valdes et al., Virology. 2009; 394:249-258). Taken together, the aforementioned results allowed selecting the aggregated form of DIIIC-2 for subsequent studies in non-human primates. The first study in non-human primates was accomplished using animals previously infected with DV2, with the main objective to know the booster capacity of DIIIC2. As expected, after administration of DIII-C2, three months after the virus infection, animals developed high levels of antiviral and neutralizing antibodies against the homologous virus, indicating the presence of functional epitopes within the recombinant protein (Valdes et al., Clin. Vaccine Immunol. 2011; 18:455-459).
As a background of this invention, it was known that addition of oligodeoxinucleotides to form aggregate variants of the protein DIIIC-2 favored the cell-mediated immunity and protection against the homologous virus in mice (Valdes et al., Virology. 2009; 394:249-258). Nevertheless, it was unknown whether the sequence can influence on the quality of the induced immune response.
According to the previous referred elements, the development of a vaccine against DV able to induce a safe and effective immune response against the four serotypes is a non-solved problem. The present invention is precisely directed to this objective.